【作者】 叶华；

【导师】 马力；

【作者基本信息】 西华大学 ， 食品科学， 2006， 硕士

【摘要】 果胶酯酶(EC:3.1.1.11)是果胶酶的一种,它是一种能催化、水解果胶生成果胶酸和甲醇的酶,英文名为pectinesterase,又称Pectin Methylesterase、Pectin demethoxylase或Pectin Methoxylase。英文简称PE或PME。国际酶委员会将它归类为果胶—果胶酰基水解酶。它存在于所有植物组织中。一些酶菌和细菌,如能引起软腐病的欧氏杆菌、干蓝黑腐黄杆菌和甘蔗流胶病黄杆菌等也能产生PE。果胶酯酶是一种广泛使用的工业酶类,主要用于果蔬饮料加工业中。目前,对果胶酯酶用于制备低酯果胶的研究也越来越受到人们的重视。对果胶酯酶基因方面的研究,国内鲜有报道,而在国外却是研究热点之一。 本文对柑橘皮中的PME进行了研究。具体研究结果如下: 1.根据核苷酸序列分析结果设计引物,利用RT-PCR技术从柑橘皮组织总RNA中扩增获得PME基因,并成功地克隆到pGEM-T easy载体上,基因全长为1797bp,开放阅读框架长1785bp,编码594个氨基酸。序列分析表明:氨基酸序列中含有5个潜在的糖基化位点,在29到48位之间可能是信号肽区域。其最大可能剪切(most likely cleavage site)位点可能在45和46位之间即:VVT-Ⅱ。大约在20-50位氨基酸之间含有一个典型的疏水性区域,这很有可能含有一个跨膜区。但是亲水性分析表明该PME序列具有高度亲水性。另外通过序列对比分析发现PME基因含有四个非常保守的Cys残基,笔者认为这个四个保守的Cys残基参与二硫桥的形成,因此与PME结构有很大关系。更多还原

【Abstract】 Pectin Methylesterase (EC:3.1.1.11),which belongs to saponifcational enzyme,can randomly hydrolyze the methyl ester bond of pectin. It is distributed in all of plants and some of microorganisms. PME is one of enzymes that widely used in industry,especially in beverage industry. Recently ,the preparation of LM-pectin by PME is more and more regarded by scholars.The research of PME gene is hotspot overseas,but fresh reports in China.PME in citrus peel has been researched in this article.The main results were as follows:1. PME gene from citrus peels was cloned using RT-PCR.We obtained a length of 1797bp gene, and its ORF is 1785bp ,coding 594 amino acid. The sequence analysis indicated that this gene has 5 potential N-glycosylation sites. Signal peptide is consisted of 19 amino acid residues,the most likely cleavage site is likely to situated in between 45 and 46(VVT-II). Hydrophobic analysis indicated that between sites 20 and 50 is a typical hydrophobic region, and it stands a good chance a span membrane region. But hydrophobic analysis indicated this PME gene is a highly hydrophile protein. PME gene has four highly conserved Cys residues, we presumed that they engaged in disulfide bridges and have a important role in PME structural.2. The clone gene is different with other PMEs from citrus sinensis in anagenesis. So may be it has a different characteristic with others. And this is happened to havethe same view that CsPME4 is a TS-PME. From phylogenetic tree we found that the similarity TS-PME is probable exist in Linum usitatissimum (flax) ,too.3. PME was abtained from citrus peels by salt saturation. Under different temperature and different pH ,the PME activity was measured by pH-stat standard method. The results were as follows: its optimal temperature was about 50°C,and under this temperature the PME has a good stability: there is still have above 80% activity after 50min heat handle at 50 °C .When above 50°C,the activity decreased obviously. Its optimal pH is about 8.A11 indicated that PME’s optimal working condition are 50°C, pH=8o更多还原